Cytotoxic ribonuclease variants

ABSTRACT

This invention relates to altered forms of members of the RNase A superfamily. An RNase A can be modified to be cytotoxic by altering its amino acid sequence so that it is not bound easily by the ribonuclease inhibitor while still retaining catalytic properties. While earlier work had identified some modifications to RNase A that would result in cytotoxicity, the use of the FADE algorithm for molecular interaction analysis has led to several other locations that were candidates for modification. Some of those modifications did result in RNase A variants with increase cytotoxicity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/559,063, filed Jul. 26, 2012 and to be issued as U.S. Pat. No.8,524,480 on Sep. 3, 2013, which is a continuation of U.S. applicationSer. No. 13/180,359, filed Jul. 11, 2011 and issued as U.S. Pat. No.8,247,190 on Aug. 21, 2012, which is a divisional of U.S. applicationSer. No. 12/177,229, filed Jul. 22, 2008 and issued as U.S. Pat. No.7,977,079 on Jul. 12, 2011, which is a divisional of U.S. applicationSer. No. 11/454,379, filed Jun. 16, 2006 and issued as U.S. Pat. No.7,416,875 on Aug. 26, 2008, which claims the benefit of U.S. provisionalApplication No. 60/690,970, filed Jun. 16, 2005. Each of theseapplications is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA073808 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Ribonucleases are enzymes that catalyze the degradation of RNA. A wellstudied ribonuclease is bovine ribonuclease A (RNase A), the putativebiological function of which it to break down the large amount of RNAthat accumulates in the ruminant gut. The RNase A superfamily is a groupof ribonuclease enzymes classified as homologous to RNase A. Some of themembers of the superfamily possess a number of interesting biologicalproperties including antiproliferative, cytotoxic, embryotoxic,aspermatogenic, and antitumoral activities. One member of this family isa homolog of RNase A originally isolated from oocytes and early embryosof the Northern leopard frog Rana pipiens, which is now known asOnconase® (ONC), a name used for the molecule which exhibits anti-tumorproperties both in vitro and in vivo. The property of degrading RNA isessential to the cytotoxicity of ONC. ONC is currently being evaluatedas a cancer therapeutic in clinical trials.

A significant limitation on the suitability of ONC as a chemotherapeuticis dose-limiting renal toxicity. ONC is retained in the kidney atconcentrations much greater than mammalian members of the RNasesuperfamily. There may also be allergenic issues with ONC, since miceproduce antibodies against ONC but not against RNase A, with which ONCshares about 30% of its amino acids. This suggests that other members ofthe RNase family may also be suitable candidates for evaluation asclinical therapeutics if they can be imbued with the cytotoxicproperties similar to ONC.

In the body, levels of RNase activity are controlled by a ribonucleaseinhibitor (RI), which is a 50-kDa protein found in the cytosol of allmammalian cells. RI is a member of a leucine rich family of proteins andis composed of 15 alternating repeats arranged symmetrically in ahorseshoe-shaped molecule. RI has a large number of cysteine residues(32 in human RI) which means that it can only keep its shape andfunction in a reducing environment like the cytosol. RI acts to bind tomembers of the RNase superfamily, one RI to one molecule of RNase, andwhen so bound, RI completely inhibits the catalytic activity of theribonuclease by steric blockage of the active site of the enzyme. Thebinding of RI to RNase is a very tight one, having a very high bindingaffinity.

Some RNase superfamily members, notably ONC and bovine seminalribonuclease, possess the native ability to evade RI. The trait ofevasion of RI is primarily responsible for the cytotoxicity of ONC andbovine seminal ribonuclease. It has also been found that RNasesuperfamily members which are not natively cytotoxic can be madecytotoxic by modifying their amino acid constituents so as to inhibitbinding to RI, and in particular, by making substitutions of largeramino acids for smaller ones at one of the points of closest interactionbetween RI and the RNase. This method is described in U.S. Pat. No.5,840,296, which describes a cytotoxic variant, G88R RNase A, which haslessened affinity for RI compared to native RNase A, but which is stillten fold less cytotoxic than ONC. The nomenclature G88R means that theRNase A molecule was altered by substituting an arginine (R) residue forthe glycine (G) residue at amino acid position 88.

The methods and tools for modeling the three-dimensional structure ofproteins continue to evolve. In analyzing the interaction between twomolecules, such as that between RNase A and RI, the problem of definingthe sites of interaction between the two molecules is only now becomingsusceptible to solution. As molecular modeling tools develop, thesophistication of the analysis of that interaction can increase.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized by an engineered ribonuclease of theRNase A superfamily having at least two amino acid changes from itsnative sequence. The first change is an amino acid substitution in theregion corresponding to amino acid residues 85 to 94 of bovinepancreatic RNase A (SEQ ID NO:1). The second change is an alteration,substitution or amino acid swap at a location selected from the groupsconsisting of an amino acid corresponding to residues 38, 39, and 67 ofbovine pancreatic RNase A.

It is an object of the present invention to define an engineeredribonuclease A that has improved cytotoxic properties compared to theprior engineered ribonucleases.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a representation of the three-dimensional structure ofribonuclease A and of ribonuclease inhibitor.

FIG. 2 is a representation of the interaction between thethree-dimensional structure of ribonuclease A and ribonuclease inhibitorshowing the sites targeted for modifications in the ribonuclease A.

FIG. 3 presents graphical data from the examples below showing theeffect of ribonucleases on the proliferation of K-562 cells. The datapoints are the means of at least three experiments each carried out intriplicate. The curves are each labeled with the corresponding variantof RNase A.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to altered ribonucleases of thesuperfamily of RNase A which have been engineered to have a new level ofcytotoxicity. This was achieved through the use of a new molecularinteraction modeling tool, the Fast Atomic Density Evaluation (FADE)algorithm. This algorithm was used to model the locations of molecularcontact between RNase A and the ribonuclease inhibitor. Based on thismodel, variants in the amino acid sequence of RNase A were designed inorder to create novel RNase A variants that through steric hindrance areable to evade the RI. These variants were also tested for ribnucleolyticactivity and for cytotoxicity. Variants are identified here that aremore cytotoxic than any previously know RNase A variants.

The analysis began with a study of the interaction between RNase A andthe RI molecule. There are many properties of a protein-proteininterface that can endow the complex with stability, including totalsurface area, nonpolar surface area, packing density, and polarinteractions. The 2,550 Å² of solvent-accessible surface area buriedupon formation of the pRI·RNase A complex is relatively large for anenzyme·inhibitor complex, and is considerably larger than the 1600 Å²that is typical for protease·inhibitor complexes. In general, proteininterfaces resemble the chemical character of solvent-exposed proteinsurfaces, which are comprised of approximately 57% nonpolar, 24% neutralpolar, and 19% charged amino acid residues. Typical protein-proteininterfaces do, however, contain fewer charged residues and more neutralpolar residues than do solvent-exposed protein surfaces. Deviating fromthis trend, the pRI-RNase A interface is significantly more charged,with 49% nonpolar, 27% neutral polar, and 24% charged residues. Indeed,electrostatics seem to play an important role in the complex formedbetween the basic Rnase A (pI 9.3) and the acidic RI protein (pI 4.7) atcytosolic pH.

In contrast to the larger role of charge-charge interactions within thepRI·RNase A complex, the degree of shape complementarity between the twosurfaces is lower than average. The shape correlation statistic, S_(c),describes how well two surfaces mesh, with a value of 1.0 describing aperfect match and 0.0 describing two unrelated surfaces. The pRI·RNase Ainterface has a relatively low S_(c) value of 0.58, as compared tovalues of 0.70-0.76 for typical protease·inhibitor complexes and0.64-0.68 for typical antibody·antigen complexes. The packing of atomsat the pRI-RNase A interface is also less dense than a typical proteininterior or protein-protein interface. The large amount of buriedsurface area could compensate for the relatively low degree of shapecomplementarity, to yield a highly stable interaction between RI andRNase A.

Prior to the work described here, K7A/K41R/G88R RNase A was the mostRI-evasive of previously produced variants. Again, under thenomenclature used here, G88R means that the RNase A molecule was alteredby substituting an arginine (R) residue for the glycine (G) residue atamino acid position 88, and the accumulation of K47A/K41R/G88R meansthat all three substitutions were made to the same RNAse A variant. Thisvariant formed a complex with pRI that had a K_(d) value of 47 nM,nearly 10²-fold greater than that of G88R RNase A. Still, K7A/K41R/G88RRNase A is not a potent cytotoxin, owing largely to the 10²-folddecrease in ribonucleolytic activity caused by the replacement of itsactive-site lysine residue with arginine. Thus to be effective as acytotoxic agent, the variant must combine reduced affinity to the RIwith the maintenance of effective catalytic activity as an RNAse.

The FADE algorithm revealed new “knobs” and “holes” in the pRI·RNase Acomplex for disruption by site-directed mutagenesis as illustrated inFIGS. 1 and 2, and listed in Table 1. In the RNase A variants created inthis work, the D38R/R39D swap and N67R substitution produced the largestdecrease in affinity for RI. Alone, each of these substitutions effecteda destabilization of the pRI·RNase A complex nearly equal to that of theG88R substitution. Combining the most disruptive FADE-inspiredsubstitutions resulted in ribonucleases that were not only 30-fold moreRI-evasive than K7A/K41R/G88R RNase A, but also retained nearlywild-type catalytic activity (see Table 2 below). Moreover, theD38R/R39D/G88R, D38R/R39D/N67R/G88R, and K31A/D38R/R39D/N67R/G88Rvariants were all more cytotoxic to K-562 cells than was ONC (see FIG.3).

The application of the FADE algorithm to the interaction of RNase A andRI thus lead to models of the interaction between the structures of thetwo molecules as illustrated in FIGS. 1 and 2. This analysis wasperformed with bovine RNase A and porcine ribonuclease inhibitor (pRI).In these figures, FADE geometric complementarity markers are displayedas solid spheres. These spheres do not represent atoms. Instead, thespheres represent points in the molecular interface near which localcomplementarity is most significant. Complementarity markers within 2 Åof any atom within a particular residue were summed to determine thecluster sizes listed in Table 1. The residues in RNase A that wereproximal to the largest number of complementarity markers and distalfrom the enzymic active site of RNase A were targeted for disruption.

One interesting finding is the difference in affinity of the porcine andhuman homologs of RI observed for some of the RNase A variants. Ingeneral, the RNase A variants were bound more tightly by pRI than hRI(data presented in Table 2 below). This higher affinity of pRI for RNaseA was not observed when the equilibrium dissociation constants weremeasured originally for complexes of wild-type RNase A with pRI(K_(d)=6.7×10⁻¹⁴ M) and hRI (K_(d)=4.4×10⁻¹⁴ M). Of the 28 pRI residuesthat contact RNase A, 25 are identical in hRI. The three differencesamong the RNase A binding residues are the replacements of His6 in pRIwith a glutamine residue in pRI, Asp228 with alanine, and Val405 withleucine. All three of these pRI residues make atom-atom contactsexclusively with FADE-identified RNase A residues. His6 makes threecontacts with Lys31 of RNase A, Asp228 makes two contacts with Ser89,and Val405 makes three contacts with Asn67. These three changes arelikely to contribute to the differential affinity of pRI and hRI for theRNase A variants.

TABLE 1 Residues of highest shape complementarity in the pRI•RNase Acomplex as identified with the FADE algorithm Chain Residue Clustersize^(a) RNase A Lys7 2 Asn24 5 Gln28 3 Lys31 2 Arg39 31 Asn67 6 Gly88 5Ser89 14 Lys91 9 RI Tyr430 22 Asp431 10 Tyr433 38 ^(a)Number of FADEcomplementarity markers within 2 Å of an atom in the indicated residue.

So these residues were the targets for potential modification. Note thevariants at the position Gly88 had previously been explored, asdescribed in U.S. Pat. No. 5,840,296, so we looked at other alterations,alone or in combination with G88R. We reasoned that disruption of theRI-RNase A complex could, in general, be achieved best by replacingsmall neutral or anionic residues in RNase A with arginine. We suspectedthat arginine, as the most polar and second largest amino acid, couldgenerate electrostatic repulsion and steric strain while increasing thenet positive charge, which is known to enhance cell internalization. Inaddition, we replaced lysine residues in RNase A with alanine to createtruncated neutral side chains and thereby eliminate favorableinteractions within the complex.

The following substitutions were thus identified as having promise forstudy:

D38R/R39D Swap (FIG. 2C). Arg39 was identified by the FADE algorithm asbeing proximal to the greatest number of complementarity markers of anyresidue in RNase A (Table 1). With 14 atom-atom contacts to pRI, Arg39also makes more contacts with RI than any residue in RNase A with theexception of Glu111, which also makes 14 contacts. Together, Asp38 andArg39 of RNase A form three hydrogen bonds with Arg453 and Glu397 ofpRI, respectively, with Arg39 interacting with Glu397 in a bidentatemanner. Additionally, these two RNase A residues make van der Waalscontacts with Gln426, Val428, Tyr430, and Ile455 of RI. Although Asp38was not identified explicitly by the FADE analysis, we reasoned that byinterchanging this residue with Arg39, we could disrupt three favorableinteractions at the pRI·RNase A interface simultaneously. Moreover, theD38R/R39D swap was conservative in that it preserved the local aminoacid content.

B4-β5 loop (FIG. 2D). Four surface loops of RNase A contribute 16 of the24 residues that contact RI. The β4-β5 loop of RNase A, containingresidues 87-96, packs against an especially hydrophobic region of pRIdefined by three tryptophan residues: Trp257, Trp259, and Trp314. ThreeRNase A residues within this loop, Gly88, Ser89, and Lys91, wereidentified by FADE as being important for mediating shapecomplementarity with RI. Earlier attempts to create an RI-evasive RNaseA showed the replacement of Gly88 (FADE cluster size 5) with an arginineresidue to be extremely effective at introducing steric andelectrostatic strain, increasing the K_(d) value of the pRI·RNase Acomplex by nearly four orders of magnitude. Therefore, although residuesSer89 and Lys91 were identified by the FADE algorithm as being nearlarge complementarity clusters, we assumed this region of the complex tobe disrupted maximally by the G88R substitution and hence did not pursuefurther alteration of this loop.

N67R substitution (FIG. 2A). Asn67 was proximal to the third largestcluster of complementarity markers, following Arg39 and residues of theβ4-β5 loop of RNase A. Asn67 makes six contacts with pRI residuesCys404, Val405, Gly406, and Tyr433, including a hydrogen bond with themain-chain oxygen of Val405. It is noteworthy that Tyr433 of pRI, whichmakes contacts with Asn67 of RNase A, was identified as being proximalto the largest number of complementarity markers of any residue ineither protein. In accordance, Tyr433 of RI and Asn67 of RNase A havebeen identified by others as the “anchor residues” in the pRI·RNase Acomplex.

Other FADE-identified residues. Lys7 (FIG. 2B) was the lowest scoring ofthe RNase A residues with a complementarity cluster size of 2.Nonetheless, Lys7 makes seven atom-atom contacts with Ser456 of pRI,including several hydrogen bonds. Previous studies had shown thisresidue to contribute significantly to complex stability. We alsoexamined Asn24 and Lys31, which make seven and four atom-atom contactsand are located near cluster sizes of 5 and 2, respectively. Asn24 makesseven van der Waals contacts and two hydrogen bonds with Asp89 andAsp117 of RI; Lys31 makes three atom-atom contacts with His6 of pRI andone contact with Asp31.

While these particular amino acid locations are identified withreference to the specific sequence of bovine pancreatic ribonuclease A,it is to be understood that there are many members of the RNase Asuperfamily of enzymes to which this information can be applied. Thoseof skill in the art today understand how to perform sequence alignmentand determine for members of this superfamily how to identify thecorresponding amino acid location in other members of this closelylinked family of enzymes.

To be successful for the purposes we envisioned, the RNase A variantshad to have reduced affinity for RI and still retain significantcatalytic activity. Hopefully, the combination of reduced affinity forRI and catalytic activity would make the variants cytotoxic and perhapsmore cytotoxic than the previous G88R RNase A variants. So modificationsof the enzyme at each of the locations identified above were tried andthe results presented in the Examples below. This work established thatseveral of these variants exhibited increased cytotoxicity. Inparticular, the variants D38R/R39D/G88R and D38R/R39D/N67R/G88R bothexhibited lowered affinity for RI, a high level of catalytic activityand both were found to have increased cytotoxicity, as exemplified bythe data presented in Tables 2 and 3 below.

We used the FADE algorithm to identify quickly and objectively RNase Aresidues within the pRI·RNase A complex that exhibit a high degree ofshape complementarity. Several of the residues identified by the FADEalgorithm were previously shown experimentally to contribute asignificant amount of binding energy to the pRI·RNase A complex or to beexcellent targets for disruption by mutagenesis. The success of the FADEalgorithm in predicting the importance of these regions gave credence toits utility and justified our subsequent analysis of additional RNase Aresidues identified by FADE. Although, a similar list of residues couldhave been identified by careful examination of the three-dimensionalstructure, the evident advantage of FADE is the extreme speed at whichit identifies regions of high complementarity. Additionally, thecomputational algorithm is objective, eliminating possible human erroror bias.

An important characteristic of any drug is its therapeutic index, whichis the ratio of its toxic dose to its effective dose. In humans, ONCexhibits a highly favorable therapeutic index as a cancerchemotherapeutic, enabling its progress to Phase III clinical trials.Mammalian ribonucleases could exhibit an even greater therapeutic indexthan does ONC. For example, the Hep-3B liver carcinoma cell line is themost vulnerable to all of the ribonucleases tested herein (Table 3). ONChas a therapeutic index (TI), here defined as IC₅₀ ^(NmuMG)/IC₅₀^(Hep-3B), of 31. In contrast, the D38R/R39D/N67R/G88R, D38R/R39D/G88R,and G88R variants of RNase A have TI values of >323, >500, and >118,respectively. A biochemical explanation for the therapeutic index ofribonucleases (amphibian or mammalian) awaits further experimentation.

EXAMPLES Experimental Protocol and Results

To explore which of the potential RNase A variants would be successful,we make expression vectors and produced each RNase A variants for eachof the propose alterations suggested above. Those variants were thantested for their catalytic properties, their affinity for RI, theirstability and their cytotoxicity.

Catalytic Activity

A ribonuclease must retain its catalytic activity to be cytotoxic.Accordingly, the catalytic activity of each ribonuclease was assayed todetermine which, if any, of the amino acid substitutions compromisedcytotoxicity by reducing the ability of the enzyme to degrade RNA.Values of k_(cat)/K_(M) for wild-type RNase A, its variants, and ONC arelisted in Table 2, which summarizes the results of this analysis. Thek_(cat)/K_(M) values of wild-type RNase A, G88R RNase A, K7A/G88R RNaseA, and ONC were 5.2×10⁷, 7.4×10⁷, 5.3×10⁶, and 2.2×10⁵ M⁻¹s⁻¹,respectively, which are in good agreement with values reportedpreviously. Swapping residues 38 and 39 of RNase A had a minor effect oncatalysis by the enzyme. The value of k_(cat)/K_(M) for D38R/R39D RNaseA was 1.8×10⁷ M⁻¹s⁻¹, which represents only a 3-fold loss inribonucleolytic activity. A similarly small effect was seen in theD38R/R39D/G88R variant; its k_(cat)/K_(M) value of 3.1×10⁷ M⁻¹s⁻¹ was2.5-fold less than that of G88R RNase A. Interestingly, when the singleR39D substitution was made in the context of the G88R substitution, theeffect on ribonucleolytic activity was more pronounced, reducing thek_(cat)/K_(M) value of G88R RNase A by 17-fold to 4.3×10⁶ M⁻¹s⁻¹. Thisdecrease could result from enhanced negative charge in this region,possibly reducing the number of productive collisions between the enzymeand its anionic substrate.

The P2 substrate binding site of RNase A, which contains Lys7, plays animportant role in catalysis by RNase A. Consistent with previousresults, K7A/G88R RNase A displayed an almost 10-fold decrease inribonucleolytic activity, having a k_(cat)/K_(M) value of 5.3×10⁶M⁻¹s⁻¹. This deleterious contribution to catalysis was additive whencombined with other amino acid substitutions that diminished activity;the D38R/R39D swap (3-fold decrease in k_(cat)/K_(M)) when combined withthe K7A substitution (10-fold decrease in k_(cat)/K_(M)) resulted in aK7A/D38R/R39D variant with an activity of 1.6×10⁶ M⁻¹s⁻¹, which is30-fold less than that of wild-type RNase A. Additionally, the K7Asubstitution was responsible for a 15-fold reduction in the activity ofD38R/R39D/G88R RNase A, reducing the activity of the quadruple variantK7A/D38R/R39D/G88R RNase A to 1.6×10⁶ M⁻¹s⁻¹.

The majority of the FADE-inspired substitutions had no significanteffect on ribonucleolytic activity. The N67R, K31A, and N24Rsubstitutions, when combined individually with the G88R substitution,produced enzymes with catalytic activity roughly comparable to that ofG88R RNase A itself. Values of k_(cat)/K_(M) for these three variantswere 9.2×10⁷, 5.2×10⁷, and 7.8×10⁷ M⁻¹s⁻¹, respectively. Therefore,RNase A variants combining many of these substitutions (such asK31A/D38R/R39D/N67R/G88R RNase A and D38R/R39D/N67R/G88R RNase A)possessed nearly the k_(cat)/K_(M) value of the wild-type enzyme(4.8×10⁷ and 3.8×10⁷ M⁻s⁻¹, respectively). In short, enzymatic activitydid not seem to be a limiting parameter for these variants.

TABLE 2 Biochemical parameters and cytotoxic activities of RNase A, itsvariants, and ONC ΔΔG^(d) T_(m) ^(a) k_(cat)/K_(M) ^(b) K_(d) (pRI)^(c)(pRI) K_(d) ^(e) (hRI) Ribonuclease (° C.) (10⁶ M⁻¹s⁻¹) (nM) (kcal/mol)(nM) wild-type Rnase A 64 52 ± 4  67 × 10^(−6h) — ND D38R/R39D RNase A60 18 ± 3  0.30 ± 0.01 5.0 — N67R RNase A 57 73 ± 19 0.36 ± 0.01 5.1 NDK7A/D38R/R39D Rnase A 62 1.6 ± 0.1 3.5 6.4 ND N24R/G88R RNase A 60 78 ±5  0.27 4.9 ND G88R RNase A 60^(i) 74 ± 4  0.57 ± 0.05^(j) 5.3 7.8^(k)K31A/G88R RNase A ND 52 ± 2  ND ND 58 ± 6  K7A/G88R Rnase A 62^(l) 5.3 ±0.4 17 ± 1  7.4 510 ± 20  R39D/G88R RNase A 61 4.3 ± 1   ND ND 6.4 ±0.3) ×10³ N67R/G88R RNase A 58 92 ± 4  45 ± 2  8.0 44 ± 7 K7A/D38R/R39D/G88R RNase A 60 1.6 ± 0.2 120 ± 10  8.5 (27 ± 3) ×10³ ONC90^(m) 0.22 ± 0.01 ≧10³ — >10³ D38R/R39D/G88R RNase A 60 31 ± 3  8.0 ±0.4 6.9 670 ± 40  K31A/D38R/R39D/N67R/G88R 54 48 ± 7  ND — (19 ± 1) ×10³RNase A D38R/R39D/N67R/G88R RNase A 56 38 ± 6  (1.4 ± 0.1) × 10³ 10.0(3.4 ± 0.1) ×10³ ND, not determined ^(a)Values of Tm (±2° C.) for RNaseA and its variants were determined in PBS by UV spectroscopy. ^(b)Valuesof k_(cat)/K_(M) (±SE) for RNase A and its variants are for catalysis of6-FAM-dArU(dA)₂-6-TAMRA cleavage at (23 ± 2) ° C. in 0.10M MES-NaOHbuffer (OVS-free) at pH 6.0, containing NaCl (0.10M). The value ofk_(cat)/K_(M) (± SE) for ONC is for catalysis of 6-FAM-dArUdGdA-6-TAMRAcleavage at (23 ± 2)° C. in 0.020M MES-NaOH buffer (OVS-free) at pH 6.0,containing NaCl (0.010M). ^(c)Values of K_(d) (±SE) are for the complexwith pRI at (23 ± 2)° C. The K_(d) value for ONC is an estimate from Wuet al., (1993) J. Biol. Chem. 268, 10686-10693. ^(d)Values of ΔΔG werecalculated with the equation: ΔΔG = −R Tln(K_(d) ^(wildtype)/K_(d)^(variant)). ^(e)Values of K_(d) (±SE) are for the complex with hRI at(23 ± 2)° C.. ^(f)Values of (k_(cat)/K_(M))_(cyto) were calculated witheq 1 and values of K_(d) for the complex with hRI. ^(g)Values of IC₅₀(±SE) are for incorporation of [methyl-³H]thymidine into the DNA ofK-562 cells exposed to a ribonuclease, and were calculated with eq 3.^(h)From Vicentini et al., (1990) Biochemistry 29, 8827-8834. ^(i)FromLeland et al., (1998) Proc. Natl. Acad. Sci. USA 95, 10407-10412.^(j)From Abel et al., (2002) Anal. Biochem. 306, 100-107. ^(k)Forfluorescein-labeled G88R RNase A. ^(l)From Haigis et al., (2002) J BiolChem 277, 11576-11581. ^(m)From Leland et al., (1998) Proc. Natl. Acad.Sci. USA 95, 10407-10412 and determined by circular dichroismspectroscopy.Affinity for Ribonuclease Inhibitor

The amino acid sequences of pRI and hRI are quite similar (77%identity). Moreover, of the 28 residues in pRI that contact RNase A,only two are replaced by dissimilar residues in hRI. Despite theassumption that the two inhibitor proteins would possess similaraffinities for the RNase A variants, we determined the K_(d) values ofcomplexes with both pRI and hRI. These K_(d) values are listed in Table2 above.

As a rigorous test of the utility of the FADE algorithm for identifyingresidues important for protein-protein interactions, we determined theK_(d) values of the FADE-inspired variants in complexes with pRI. TheK_(d) values of 0.57 and 17 nM obtained for G88R RNase A and K7A/G88RRNase A in complexes with pRI, were in good agreement with thosedetermined previously. The N24R substitution was the only change thatdid not diminish the affinity of pRI for RNase A. Indeed, with a K_(d)value of 0.27 nM, N24R/G88R RNase A actually appeared to form a slightlytighter complex with pRI than did G88R RNase A. The most significantincreases in values of K_(d) were observed for the D38R/R39D swap andthe N67R substitution, whose complexes exhibited K_(d) values of 0.30and 0.36 nM, respectively. These amino acid changes were responsible for4,500- and 5,400-fold increases in K_(d) value, respectively. TheK7A/D38R/R39D, N67R/G88R, and D38R/R39D/G88R variants formed complexeswith pRI that have K_(d) values of 3.5, 45, and 8.0 nM, respectively.

The combination of multiple substitutions produced the most RI-evasivevariants of RNase A. Of note are the K7A/D38R/R39D/G88R andD38R/R39D/N67R/G88R variants, which formed complexes with pRI havingK_(d) values of 0.12 and 1.4 μM, respectively. Notably,D38R/R39D/N67R/G88R RNase A is the first RNase A variant observed toform a complex with pRI that has a micromolar K_(d) value. By changingonly four out of 124 residues in RNase A, the K_(d) value of thepRI·RNase A complex was increased by 20-million fold with theD38R/R39D/N67R/G88R variant.

Values of K_(d) for the complexes of pRI with RNase A variants are idealfor assessing the ability of the FADE algorithm to identifyshape-complementarity markers. As a chemotherapeutic, however, cytotoxicribonucleases must be capable of eluding human RI. For this reason,values of K_(d) were also determined for the hRI complexes with RNase Avariants. With the exception of N67R/G88R RNase A (K_(d)=44 nM), K_(d)values for the hRI complexes were greater than those obtained for pRI,with the magnitude of the differences ranged from 2- to 230-fold. Thehighest K_(d) value observed for a complex with hRI was that ofK7A/D38R/R39D/G88R RNase A at 27 μM, which represents a 400 million-folddecrease in affinity for hRI.

Importantly, the destabilizing effects of these substitutions on thecomplex were not entirely additive, indicating that the pRI·RNase Ainterface is plastic. The accommodating nature of the binding interfacecan be seen upon comparison of ΔΔG values (Table 2). For example, theG88R and N67R substitutions destabilized the complex by approximately 5kcal/mol each. Yet, the N67R/G88R double variant exhibited an 8 kcal/molloss in binding free energy, despite the spatial separation of these twosubstitutions.

Stability

The conformational stability of a ribonuclease is necessary forbiological function, including cytotoxicity. Hence, the T_(m) value ofeach RNase A variant was determined and is listed in Table 2. The N67Rsubstitution was the most destabilizing, decreasing the T_(m) value ofwild-type RNase A by 7° C. to a value of 57° C. This loss inconformational stability was not recovered by additional substitutions,being observed in all variants containing the N67R substitution. TheN67R/G88R and D38R/R39D/N67R/G88R variants had T_(m) values of 58 and 56C, respectively. K31A/D38R/R39D/N67R/G88R RNase A had the lowest T_(m)value of 54° C., which is nearly 10 C lower than that of the wild-typeenzyme. Still, this T_(m) value is significantly greater thanphysiological temperature. None of the other amino acid substitutionsreduced the T_(m) value by more than a few degrees C.

Cytotoxicity

The toxicity of each ribonuclease was measured with the K-562 humanleukemia cell line. Ribonucleases are listed in order of increasingcytotoxicity in Table 2, using IC₅₀ values derived by applying equation3 (set forth below) to the data in FIG. 3 (h=1.43±0.02 for the 12cytotoxic ribonucleases). ONC, G88R RNase A, and K7A/G88R RNase Adisplayed IC₅₀ values similar to those reported previously. D38R/R39DRNase A (FIG. 3A) and N67R RNase A (FIG. 3C) exhibited no cytotoxicactivity, even at concentrations of 25 μM. The lack of cytotoxicity forthe latter two variants is interesting, considering the large increasein cytotoxicity they exhibited in the context of the G88R substitution.

Upon incorporation of the K7A substitution into the D38R/R39D/G88Rvariant, its affinity for hRI decreased 40-fold, consistent with theloss of favorable interactions between the lysine side chain andC-terminal serine residue of hRI. This larger K_(d) value wasaccompanied by a loss in catalytic activity, leading to an IC₅₀ valuenearly twice that of D38R/R39D/G88R RNase A. Although Asp38 was notidentified explicitly by the FADE analysis, its importance in theconservative D38R/R39D swap is apparent when the IC₅₀ value of R39D/G88RRNase A (IC₅₀=0.69 μM) is compared with that of D38R/R39D/G88R RNase A(IC₅₀=0.22 μM).

Two of the most cytotoxic variants of RNase A discovered in this work,D38R/R39D/G88R and D38R/R39D/N67R/G88R, as well as ONC, wild-type RNaseA, and G88R RNase A, were screened for cytotoxic activity against tendifferent cell lines. The resulting IC₅₀ values of these ribonucleasesare listed in Table 3. All of the cell lines are of human origin exceptfor NmuMG, which is a mouse mammary normal epithelial cell line. Withthe exception of the Hep3B cell line, all of the human cancer celllines, like the human leukemia K-562 line, are among the 60 cell linesscreened by the National Cancer Institute in search of novel cancerchemotherapeutics.

The cell lines are listed in Table 3 according to increasing doublingtimes. There did not appear to be any direct correlation betweendoubling time and sensitivity to the ribonucleases as had been reportedpreviously. In general, the trend of cytotoxicity among the RNase Avariants reflected that seen in the K-562 cell line, namelyD38R/R39D/N67R/G88R>D38R/R39D/G88R >G88R >wild-type RNase A, with theD38R/R39D/N67R/G88R variant consistently having the lowest IC₅₀ value.The HCT-116, A549, and SF268 cell lines were exceptions to this generaltrend, as all were more sensitive to wild-type RNase A than was G88RRNase A. Others have reported that wild-type human pancreaticribonuclease (RNase 1) is toxic to some cell lines, just as we foundseveral cell lines susceptible to wild-type RNase A. These three celllines derive from three different tissue types: colon, lung, and CNS,respectively.

A goal of this work was to identify RNase A variants possessingcytotoxicity equal to or greater than that of ONC. This goal wasachieved with the D38R/R39D/N67R/G88R variant in the K-562, Dul45,Hep-3B, and SF268 cell lines. In the remaining six cell lines, ONCexhibited 3- to 30-fold greater cytotoxicity than did the RNase Avariants. Interestingly, none of the RNase A-derived variants tested inthis screen was toxic to the normal cell line NmuMG at the maximumconcentrations tested. This discrimination was not observed with ONC,which had an IC₅₀ of 1.62 μM for the normal mouse cell line.

TABLE 3 IC₅₀ values of RNase A, its variants, and ONC for ten cell linesIC₅₀ (μM)^(a) Doubling wild- Cell line Description time (h) type DRNGbDRGb G88R ONC HCT-116 colon 17.4 4.7 0.49 1.4 10.4 0.14 carcinomaNCI-H460 lung carcinoma 17.8 39 0.71 0.60 11.0 0.13 A549 lung 22.9 15.54.8 13.7 27.0 0.15 adenocarcinoma MCF-7 breast 25.4 21.7 0.27 0.42 4.40.086 adenocarcinoma Du145 prostate 32.3 5.5 0.085 0.45 2.0 0.11carcinoma SF-268 CNS 33.1 3.8 0.18 0.64 4.6 0.088 glioblastoma NCI/ADR-breast 34.0 19 1.00 2.3 5.8 0.06 RES adenocarcinoma SK-OV-3 ovary 48.73.2 0.76 1.5 2.8 0.13 adenocarcinoma Hep-3B liver carcinoma ND 2.8 0.0310.040 0.34 0.051 NmuMG mammary ND >40 >10 >20 >40 1.6 normal epithelial(mouse) ^(a)Values of IC₅₀ are for the conversion of calcein AM tocalcein in cells exposed to a ribonuclease, and were calculated with eq4. ^(b)DRNG and DRG refer to the D38R/R39D/N67R/G88R and D38R/R39D/G88Rvariants of RNase A, respectively.

Methods And Materials

Materials

Escherichia coli BL21(DE3) cells and pET22b(+) and pET27b(+) plasmidswere from Novagen (Madison, Wis.). K-562 cells were derived from acontinuous human chronic myelogenous leukemia line obtained from theAmerican Type Culture Collection (Manassas, Va.). Cell culture mediumand supplements were from Invitrogen (Carlsbad, Calif.).[methyl-³H]Thymidine (6.7 Ci/mmol) was from Perkin Elmer (Boston,Mass.). Enzymes were obtained from Promega (Madison, Wis.) or NewEngland Biolabs (Beverly, Mass.). Ribonuclease substrates6-FAM-dArUdAdA-6-TAMRA and 6-FAM˜dArUdGdA˜6-TAMRA were from IntegratedDNA Technologies (Coralville, Iowa). All other chemicals used were ofcommercial reagent grade or better, and were used without furtherpurification.

Terrific Broth (TB) contained (in 1.00 L) tryptone (12 g), yeast extract(24 g), glycerol (4 mL), KH₂PO₄ (2.31 g), and K₂HPO₄ (12.54 g).Phosphate-buffered saline (PBS) contained (in 1.00 L) NaCl (8.0 g), KCl(2.0 g), Na₂HPO₄.7H₂O (1.15 g), KH₂PO₄ (2.0 g), and NaN₃ (0.10 g), andhad pH 7.4.

Instruments

[methyl-³H]Thymidine incorporation into K-562 genomic DNA wasquantitated by scintillation counting using a Microbeta TriLux liquidscintillation and luminescence counter (Perkin Elmer, Wellesley, Mass.).The mass of each protein variants was confirmed by MALDI-TOF massspectrometry using a Voyager-DE-PRO Biospectrometry Workstation (AppliedBiosystems, Foster City, Calif.). Fluorescence measurements were madewith a QuantaMaster1 photon-counting fluorometer equipped with samplestirring (Photon Technology International, South Brunswick, N.J.).Thermal denaturation data were acquired using a Cary 3 double-beamspectrophotometer equipped with a Cary temperature controller (Varian,Palo Alto, Calif.).

Design of Ribonuclease A Variants

The Fast Atomic Density Evaluator (FADE) program calculatesshape-complementarity markers of proteins at complex interfaces. Atomicdensity is measured using fast Fourier transform algorithms based onmethods described previously. Using the structure of the crystallinepRI·RNase A complex (PDB entry IDFJ), critical RNase A residues in closeproximity to large clusters of shape-complementarity markers wereidentified and are listed in Table 1 above Amino acid substitutions werechosen to create maximal electrostatic or steric conflict as well aseliminate any favorable Coulombic or short-range interactions.

At the onset of this research, the most cytotoxic variant of RNase'Aknown was K7A/G88R RNase A. Subsequent amino acid substitutions inspiredby FADE analysis were initially made in the background of theseestablished changes, with the expectation that any additionalcontributions to evasivity would be additive. As discussed here, wefound that the loss of enzymatic activity accompanying the K7Asubstitution compromised cytotoxicity, and hence, later substitutionswere made in the background of the G88R substitution alone. The G88Rbackground provided a well-characterized benchmark of cytotoxicity andRI-evasion from which we could identify improvements using ourestablished assays.

Substitutions that were Successful in the G88R Background were also madeAlone to Assess their Individual Contribution to Evasion of RI andCytotoxicity.

Production of Ribonucleases

cDNA molecules encoding RNase A variants were created byoligonucleotide-mediated site-directed mutagenesis using a pET22b(+) orpET27b(+) plasmid that contained cDNA encoding wild-type RNase A or itsG88R variant, respectively. ONC, wild-type RNase A, and RNase A variantswere produced as described previously in Haigis et al., (2002) J BiolChem 277, 11576-11581, with the following exceptions. Inclusion bodiesfrom E. coli were stirred in 20 mM Tris-HCl buffer at pH 8.0, containingguanidine-HCl (7 M), DTT (0.1 M), and EDTA (10 mM) until dissolvedthoroughly. Ribonucleases were refolded overnight at room temperaturefollowing slow dilution into 0.10 M Tris-HCl buffer at pH 8.0,containing NaCl (0.1 M), reduced glutathione (1.0 mM), and oxidizedglutathione (0.2 mM). Following purification, proteins were dialyzedagainst PBS and filtered with a 0.2 μm syringe prior to use inbiochemical assays. Protein concentration was determined by UVspectroscopy using an extinction coefficient of E₂₇₈=0.72 mg·ml⁻¹cm⁻¹for RNase A and its variants and ε₂₈₀=0.87 mg·ml⁻¹cm⁻¹ for ONC.

Production of Ribonuclease Inhibitor

Porcine ribonuclease inhibitor (pRI) was prepared as described in Klinket al., (2001) Protein Expr. Purif. 22, 174-179. Freshly prepared pRIwas confirmed to be 100% active by its ability to titrate theribonucleolytic activity of wild-type RNase A.

Human ribonuclease inhibitor (hRI) was produced in E. coli BL21(DE3)cells transformed with a pET22b(+) plasmid that contained cDNA encodinghRI between its NdeI and SalI sites. Cultures (1.0 L) of TB wereinoculated to an OD of 0.005 at 600 nm from an overnight culture. Theculture was grown at 37 C to an OD of 1.8-2.0 at 600 nm. IPTG was addedto a final concentration of 0.5 mM, and induction was carried outovernight at 18 C. Subsequent purification of soluble protein andactivity determination of hRI was carried out in the same manner as forpRI. The purity and size of both RIs were confirmed by electrophoresisand mass spectrometry.

Assays of Ribonuclease Inhibitor Binding

The affinity of RNase A variants for both pRI and hRI was determined byusing a slight modification of a competition assay reported previouslyin Abel et al., (2002) Anal. Biochem. 306, 100-107. Briefly, bothfluorescein-labeled G88R RNase A (final concentration: 50 nM) andvarying concentrations of an unlabeled ribonuclease were added to 2.0 mlof PBS containing DTT (5 mM). Following a 15 min incubation at (23±2)°C., protected from light, the initial fluorescence intensity of theunbound fluorescein-labeled G88R RNase A was monitored for 3 min(excitation: 493 nm, emission: 515 nm). pRI was then added (finalconcentration: 50 nM, which is sufficient to bind 90% of thefluorescein-labeled G88R RNase A in the absence of unlabeledcompetitor), and the final fluorescence intensity was measured. Thecompetition assay was carried out identically for hRI, except that morehRI was necessary (final concentration of 115 nM) to achieve 90% bindingof fluorescein-labeled G88R RNase A because of the lower affinity ofhRI. The affinity of hRI for fluorescein-labeled G88R RNase A wasdetermined by titrating 50 nM fluorescein-labeled G88R RNase A withvarying amounts of hRI (0.5-1000 nM) and recording the decrease influorescence upon binding. The value of K_(a) was found to be 7.8 nM.

Assays of Catalytic Activity

The ribonucleolytic activities of RNase A and its variants weredetermined by assaying their ability to cleave the hypersensitivefluorogenic substrate 6-FAM˜dArUdAdA˜6-TAMRA (50 nM), which exhibits a180-fold increase in fluorescence (excitation: 493 nm, emission: 515 nm)upon cleavage. Assays were carried out at (23±2)° C. in 2.0 ml of 0.10 MMES-NaOH buffer at pH 6.0, containing NaCl (0.10 M). The MES used toprepare the assay buffer was purified by anion-exchange chromatographyto remove trace amounts of oligomeric vinylsulfonic acid, which is abyproduct of commercial buffer synthesis and has been shown to be apotent inhibitor of RNase A. Values of k_(cat)/K_(M) were obtained withthe equation:

$\begin{matrix}{{k_{cat}/K_{M}} = {\left( \frac{\Delta\;{I/\Delta}\; t}{I_{\max} - I_{0}} \right)\frac{1}{\lbrack{ribonuclease}\rbrack}}} & (1)\end{matrix}$

where ΔI/Δt represents the initial reaction velocity generated bycleavage of the 6-FAM-dArUdAdA-6-TAMRA substrate upon addition ofribonuclease to the cuvette. I₀ and I_(max) are, respectively, thefluorescence intensities prior to enzyme addition and following thecomplete cleavage of substrate by excess wild-type RNase A. Activityvalues for ONC were determined at (23±2) C in 2.0 ml of OVS-free 20 mMMES-NaOH buffer at pH 6.0, containing NaCl (0.010 M) using the substrate6-FAM˜dArUdGdA˜6-TAMRA (50 nM).

Assays of Cytotoxicity

IC₅₀ values for RNase A, its variants, and ONC were determined bymeasuring the incorporation of [methyl-³H]thymidine into the cellularDNA of K-562 cells in the presence of ribonucleases. All cytotoxicityassays were repeated at least three times in triplicate. Each data pointrepresents the mean of three or more experimental values (±SE). IC₅₀values were calculated by fitting the curves using nonlinear regressionto a sigmoidal Dose-response curve with the equation:

$\begin{matrix}{y = \frac{100\%}{1 + 10^{{({{\log{({IC}_{50})}} - {\log{\lbrack{ribonuclease}\rbrack}}})}h}}} & (2)\end{matrix}$

In eq 3, y is the total DNA synthesis following a 4-h[methyl-³H]thymidine pulse, and h is the slope of the curve.

Cytotoxicity assays other than those carried out using K-562 cells wereperformed at the Keck-UWCCC Small Molecule Screening Facility. Theseassays used ten cell lines from a broad spectrum of tissues. Following a72-h incubation with ribonucleases, IC₅₀ values were determined bymeasuring the enzymatic conversion of the profluorophore calcein AM(Molecular Probes, Eugene, Oreg.) to calcein in live cells. Coefficientof variation and Z-scores were determined for each cell line usingdoxorubicin as an internal control. All cytotoxicity assays wereperformed in triplicate three times. IC₅₀ values were calculated withthe equation:

$\begin{matrix}{{IC}_{50} = {{\left( \frac{{50\%} - {{low}\mspace{14mu}\%}}{{{high}\mspace{14mu}\%} - {{low}\mspace{14mu}\%}} \right)\left( {\lbrack{ribonuclease}\rbrack_{high} - \lbrack{ribonuclease}\rbrack_{low}} \right)} + \lbrack{ribonuclease}\rbrack_{low}}} & (3)\end{matrix}$

where low % and high % refer to inhibition by the two concentrations,[ribonuclease]_(low) and [ribonuclease]_(high), that bracket 50%inhibition.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is understood that certain adaptations of theinvention are a matter of routine optimization for those skilled in theart, and can be implemented without departing from the spirit of theinvention, or the scope of the appended claims.

We claim:
 1. A method for producing a modified cytotoxic ribonuclease,comprising: a) producing a cytotoxic ribonuclease from cells transformedwith an expression vector encoding a modified cytotoxic ribonuclease;and b) purifying said modified cytotoxic ribonuclease; wherein saidmodified cytotoxic ribonuclease comprises an amino acid sequence whichdiffers from the amino acid sequence of SEQ ID NO:1 solely by four toseven amino acid substitutions, wherein said four to seven amino acidsubstitutions are located at positions of SEQ ID NO:1 selected from thegroup consisting of 7, 31, 38, 39, 67 and 85-94, wherein at least one ofthe four to seven amino acid substitutions is at a positioncorresponding to any one of positions 85-94 of SEQ ID NO: 1, wherein atleast three of the four to seven amino acid substitutions are atpositions corresponding to positions 7, 31, 38, 39, or 67 of SEQ IDNO:1.
 2. The method of claim 1, wherein said cells are E. coli cells. 3.The method of claim 2, wherein said purifying comprises treatinginclusion bodies from E. coli with a buffer solution until dissolvedthoroughly.
 4. The method of claim 1, wherein said purifying comprisesdialyzing said modified cytotoxic ribonuclease against PBS buffer. 5.The method of claim 4, further comprising the step of filtering thedialyzed modified cytotoxic ribonuclease.
 6. The method of claim 1,further comprising the step of determining protein concentration of thepurified modified cytotoxic ribonuclease.
 7. A method for producing amodified cytotoxic ribonuclease, comprising: a) producing a cytotoxicribonuclease from cells transformed with an expression vector encoding amodified cytotoxic ribonuclease; and b) purifying said modifiedcytotoxic ribonuclease; wherein said modified cytotoxic ribonucleasecomprises an amino acid sequence which differs from the amino acidsequence of SEQ ID NO: 1 solely by three or more amino acidsubstitutions at positions corresponding to positions of SEQ ID NO:1selected from the group consisting of 38, 39, 67 and 85-94, wherein atleast one of the three or more amino acid substitutions is at a positioncorresponding to any one of positions 85-94 of SEQ ID NO:1, wherein atleast two of the three or more amino acid substitutions are at positionscorresponding to positions 38, 39 or 67 of SEQ ID NO:1.
 8. The method ofclaim 7, wherein said cells are E. coli cells.
 9. The method of claim 8,wherein said purifying comprises treating inclusion bodies from E. coliwith a buffer solution until dissolved thoroughly.
 10. The method ofclaim 7, wherein said purifying comprises dialyzing said modifiedcytotoxic ribonuclease against PBS buffer.
 11. The method of claim 10,further comprising the step of filtering the dialyzed modified cytotoxicribonuclease.
 12. The method of claim 7, further comprising the step ofdetermining protein concentration of the purified modified cytotoxicribonuclease.
 13. A method for producing a modified cytotoxicribonuclease, comprising: a) producing a cytotoxic ribonuclease fromcells transformed with an expression vector encoding a modifiedcytotoxic ribonuclease; and b) purifying said modified cytotoxicribonuclease; wherein said modified cytotoxic ribonuclease comprises anamino acid sequence which differs from the amino acid sequence of SEQ IDNO:1 solely by two or more modifications at positions corresponding topositions of SEQ ID NO:1 selected from the group consisting of 38, 39,67 and 85-94, wherein at least one of the two or more modifications isan amino acid substitution at a position corresponding to any one ofpositions 85-94 of SEQ ID NO:1, and wherein at least one of the two ormore modifications is selected from the group consisting of: (i) asubstitution at a position corresponding to position 39 of SEQ ID NO: 1,(ii) a substitution at a position corresponding to position 67 of SEQ IDNO: 1, (iii) substitutions at positions corresponding to positions 38and 39 of SEQ ID NO: 1, and (iv) substitutions at positionscorresponding to positions 38, 39 and 67 of SEQ ID NO:
 1. 14. The methodof claim 13, wherein said cells are E. coli cells.
 15. The method ofclaim 14, wherein said purifying comprises treating inclusion bodiesfrom E. coli with a buffer solution until dissolved thoroughly.
 16. Themethod of claim 13, wherein said purifying comprises dialyzing saidmodified cytotoxic ribonuclease against PBS buffer.
 17. The method ofclaim 16, further comprising the step of filtering the dialyzed modifiedcytotoxic ribonuclease.
 18. The method of claim 13, further comprisingthe step of determining protein concentration of the purified modifiedcytotoxic ribonuclease.